Fig 1.
garA is required for growth of M. tuberculosis in vitro, survival in macrophages, and virulence in mice.
(A) M. tuberculosis lacking garA was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. (B) M. tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M. tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. (C) M. tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 105 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with ΔgarAMt (red squares) was significantly lower than those infected with M. tuberculosis H37Rv (black circles), or complemented ΔgarAMt (black triangles) at all time points from day 7 (p<0.005, t test). (D) M. tuberculosis lacking garA failed to disseminate to the spleen by day 28 (symbols match panel C). The minimum number of bacteria that could be detected was 45 CFU/organ, marked by a dashed black line in C and D.
Fig 2.
Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M. smegmatis.
(A) All strains grew at the same rate on standard Middlebrook 7H9 medium. (B) ΔgarAMs grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH4Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA). (C + D) ΔpknGMs grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon (C) or nitrogen source (D) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. (E) ΔpknGMs formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve (D) imaged at 60 hours. Growth of ΔgarAMs complemented with phosphoablative GarA (EAAS) was equivalent to that of ΔpknGMs complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.
Fig 3.
pknG disruption in M. tuberculosis caused a nutrient-dependent growth defect.
(A) All strains grew at the same rate on minimal medium supplemented with glycerol 0.2%. ΔpknGMt (blue diamonds) grew more slowly than wild type M. tuberculosis (black circles) when the sole carbon source was (B) glutamate 10 mM or (C) asparagine 10 mM. Plasmid-encoded PknG (empty circles) partially restored the growth defect. Graphs show the mean and standard deviation of three independent experiments.
Fig 4.
Investigation of GarA phosphorylation in M. smegmatis and M. tuberculosis.
(A) Addition of Phos-tag reagent to SDS-PAGE allowed separation of phosphorylated GarA (GarA-P) from GarA. Cell extracts were prepared from M. smegmatis and M. tuberculosis wild type and pknG deletion strains and complemented strains. (B) The same cell extracts of M. tuberculosis wild type and pknG deletion strain were analysed by LC-MS/MS to detect the tryptic peptide of GarA carrying the ETTS phosphorylation motif. The abundance of the peptide with no phosphorylation is shown in black and the T21-phosphorylated peptide is shown in red. A peptide from another part of the protein was also measured as a control (blue). (C) A reporter strain of ΔgarAMs carrying plasmids encoding hexahistidine-tagged garA, or variants of garA, confirmed that most phosphorylation occurred at the first threonine in the ETTS motif. GarA from cell lysates was visualised by Western blotting. Images shown are representative of three or more independent replicates.
Fig 5.
GarA phosphorylation in M. smegmatis was regulated by nutrient availability.
The reporter strain of M. smegmatis was cultured in different media and cell lysates analysed by Western blot and densitometry. (A) Glutamate and related amino acids triggered phosphorylation of GarA: the nitrogen source is indicated and the carbon source was glucose. (B) The supplied carbon source affected phosphorylation of GarA: the carbon source is indicated and the nitrogen source was NH4Cl. (C) Phosphorylation of GarA occurred rapidly when cells were cultured in poor medium and then given supplementary nutrients (initially 1% glucose with 10 mM NH4Cl and 0.05% tyloxapol, then 1% v/v glycerol and 30 mM asparagine were added at time zero). (D) Dephosphorylation of GarA occurred slowly when cells switched from rich to poor medium (initially 1% glycerol with 30 mM asparagine and 0.05% Tween 80 then switched to 1% glucose with 10 mM NH4Cl and 0.05% tyloxapol). (E) GarA was predominantly unphosphorylated when M. smegmatis were in stationary phase or starved in PBS. The reporter strain of M. smegmatis was grown in Sauton’s medium with shaking for 5 days. For the starvation experiment exponentially growing M. smegmatis were washed with PBS and incubated in PBS with 0.05% tyloxapol for 5 days. Values represent mean and standard deviation of at least three independent replicates.
Fig 6.
GarA was required during stationary phase for the maintenance of intracellular glutamate and for survival.
(A) M. smegmatis lacking garA failed to maintain intracellular glutamate and glutamine pools during extended stationary phase in 7H9 medium. Intracellular glutamate and glutamine were measured for wild type M. smegmatis (black circles), ΔgarAMs (red squares), and complemented ΔgarAMs (black triangles). Inset graphs show intracellular metabolites for the same experiment at day 28 (B) M. smegmatis lacking garA gradually lost viability during prolonged stationary phase. Cells were cultured in 7H9 medium over a time period of five months. Aliquots were withdrawn at regular intervals and surviving cells were plated on 7H10 to calculate CFU ml-1. All experiments were repeated at least 3 times and data show the mean with standard deviation.
Fig 7.
Deletion of garA or disruption of GarA phosphorylation caused changes in intracellular glutamate and other changes in the metabolome of M. smegmatis.
Intracellular metabolites from wild type M. smegmatis and mutants were analysed by mass spectrometry using an untargeted metabolomics approach. M. smegmatis lacking garA has lower intracellular glutamate and metabolites related to glutamate than the parental strain (A) but plasmid encoded GarA reversed this change (B). M. smegmatis expressing truncated GarA that lacks phosphorylation sites had higher intracellular ornithine than wild type (C). Intracellular glutamate concentrations for the mutant strains are compared to wild type in panel (D), together with those metabolites that were significantly changed in >1 mutant strain but not in complemented strains. pSer is O-phosphoserine, Orn is ornithine, Cit is citrulline, MaltoP is maltopentaose. (A-C) Each point on the scatter plots represents a single metabolite. Metabolites with the greatest fold-change and statistical significance are highlighted (log2(fold change)>0.5 and q-value<0.05), thresholds marked as dashed lines on graphs): metabolites at lower concentration are blue and those at higher concentration are red. These data were taken from cells growing in Middlebrook 7H9 in early exponential phase and represent the mean from at least 3 independent experiments.
Table 1.
Summary comparing the intracellular metabolome of mutant strains with parental M. smegmatis.
Metabolites that are significantly changed in more than one strain are in bold type. Metabolites related to amino acid metabolism are highlighted with asterisks *. Metabolites are listed in order of fold-change compared to M. smegmatis, and those that were >2-fold changed are shaded.
Fig 8.
A model depicting control of the TCA cycle and glutamate metabolism by PknG and GarA.
When PknG activity is low, unphosphorylated GarA activates glutamate synthesis and inhibits glutamate catabolism by direct binding to the relevant enzymes. When PknG activity is stimulated, for example by glutamate and aspartate, GarA become phosphorylated, causing a shift in metabolism towards glutamate catabolism. KDH is the alpha-ketoglutarate dehydrogenase complex, GDH is glutamate dehydrogenase, GltS is glutamate synthase.
Table 2.
Precursor/Product ion transition used for multiple reaction monitoring of GarA peptides.